Project Summary/Abstract Tumors continuously interact with their surroundings, using mechanosensitive proteins to detect and respond to the microenvironment. PIEZO1 is a mechanosensing ion channel that is activated by cell membrane tension. In epithelial monolayers, PIEZO1 is required for live cell extrusion in areas of overcrowding, while promoting proliferation in response to cell stretching or low cell density. PIEZO1 relocalizes from the plasma membrane to the cytoplasm with increasing cell density, suggesting that its dichotomous effects on proliferation or extrusion are dictated by its localization. Of note, breast cancer cells have been shown to express functional PIEZO1 channels. However, studies to date have not interrogated the function or localization of PIEZO1 in three- dimensional (3D) systems, and the relevance of PIEZO1-mediated proliferation and extrusion for cancer progression is unclear. In my preliminary work, I have found that high PIEZO1 expression is associated with poorer overall survival in meta-analysis of breast cancer cohorts, even when controlling for tumor grade or molecular subtype. In addition, I have found that PIEZO1 is more highly expressed in metastases compared to primary breast tumors, and across diverse cancer types, I observed that PIEZO1 expression is associated with focal adhesion, integrin signaling, and cell motility pathways. Collectively, these findings implicate PIEZO1 as a mechanosensor that promotes cancer progression by relocalizing in response to microenvironmental stimuli to drive the proliferation or extrusion of tumor cells. I hypothesize that PIEZO1 is a dynamic sensor of mechanical forces in 3D systems, driving breast cancer tumorigenesis and metastasis. My first aim is to assess the effect of cell density and substrate stiffness on PIEZO1 localization in developing breast cancer spheroids. To accomplish this, I will generate MCF-7 human breast cancer cell lines expressing GFP-tagged endogenous PIEZO1 (PIEZO1-GFP), as well as H2B-mCherry and LifeAct-tagBFP2 reporters. I will utilize live confocal imaging to quantify the cellular localization of PIEZO1 as MCF-7 cells proliferate to form spheroids in 3D culture. Additionally, I will assess PIEZO1 localization in MCF-7 spheroids grown in environments of varying stiffness. My second aim is to interrogate the effect of PIEZO1 loss on breast cancer progression. To do so, I have generated PIEZO1-knockout (KO) MCF-7 cells and Piezo1-KO E0771 murine breast cancer cells. I will assess the spheroid forming ability of KO vs. control MCF-7 cells grown in 3D culture. I will also orthotopically inject KO vs. control E0771 cells into mice to compare their tumorigenic and metastatic capacity. Building on my preliminary computational work, I will further investigate whether PIEZO1-KO leads to aberrations in focal adhesion-integrin signaling in breast cancer spheroids. If successful, this project will establish that PIEZO1 dynamically localizes in response to mechanical stimuli in 3D systems, and will demonstrate a functional role for PIEZO1 in breast cancer progression.